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We describe the in vivo emergence of resistance to ceftazidime/avibactam via modification of AmpC in a clinical Klebsiella aerogenes isolate during therapy with this combination. Paired ceftazidime/avibactam-susceptible/resistant isolates were obtained before and during ceftazidime/avibactam treatment. Whole genome sequencing revealed a differential mutation in AmpC (R148W) in the ceftazidime/avibactam-resistant isolate. Molecular cloning and structural studies confirmed the impact of this substitution, which affects the architecture of the H10 helix, on the evolved resistant phenotype., Competing Interests: G.B. has received funding and study materials from MSD, grants contracts from MSD, Pfizer, ABAC Therapeutics, and Roche, consulting fees and honoraria for lectures and/or presentations from MSD, Shionogi Pfizer, Roche, and Menarini, and support for attending meetings and/or travels from Pfizer. J.A.-S. has received honoraria for lectures, consulting fees, and/or presentations from MSD, Shionogi, Pfizer, and Advanz Pharma.

We report the isolation of a bla OXA-181 -positive, tigecycline-resistant Klebsiella aerogenes strain KA04 from a Chinese inpatient's fecal sample. Species identification was performed using MALDI-TOF MS. The antibiotic susceptibilities were assessed via the broth microdilution method. To elucidate the transmission and genetic structure of the bla OXA-181 gene, conjugation assays and whole-genome sequencing (WGS) were performed. KA04 displayed resistance to carbapenems, quinolones, piperacillin/tazobactam and tigecycline. Through WGS and conjugation experiments, it was possible to confirm bla OXA-181 and qnrS1 genes causing antibiotic resistance were located on a 51-kb IncX3 type mobile plasmid, bla OXA-181 gene could be successfully transferred into E. coli EC600 at a conjugation frequency of 1.1 × 10 - 4 . tet(A) gene was located on both the chromosome and non-transmissible IncFIB(K) plasmid. This is a tigecycline-resistant K. aerogenes harboring bla OXA-181 isolate from human fecal sample, highlighting a significant public health concern. Further comprehensive surveillance is needed., Competing Interests: Declarations. Ethical approval: Not required. Competing interests: None declared., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The fitness cost associated with antimicrobial resistance has an important influence on evolutionary dynamics. We compared the genomes of three Klebsiella aerogenes isolates recovered from blood samples or deep abscess cultures from the same patient: the wild-type strain (CT_WT), a piperacillin-tazobactam-resistant strain (CT_PENI), and an extended-spectrum-cephalosporin (ESC)-resistant strain (CT_R). Whole-genome sequencing revealed that CT_PENI had acquired a TEM-1 β-lactamase with a mutated promoter, accounting for overproduction. CT_PENI then acquired an E240G substitution in the TEM-1 β-lactamase (resulting in TEM-207) and lost the porin-encoding ompK36 gene to give CT_R. All three strains showed the same virulence in a mouse model of intraperitoneal infection. The results of recombination and transformation assays indicated that when present separately, the TEM-207 overproduction and the ompK36 gene deletion had only small effects on susceptibility to ESCs. However, the combination of the two changes led to a much lower susceptibility to ESCs. Moreover, the levels of fitness in vitro and in vivo in a murine model of gut colonization were significantly lower after TEM-1 β-lactamase overproduction and lower still after E240G substitution and OmpK36 loss. We hypothesize that the chosen courses of antibiotics led to the stepwise emergence of a clone with resistance to penicillins and ESCs and no loss of virulence. However, acquired resistance may have a fitness cost that limits evolutionary success. Our results might explain why the overproduction of extended-spectrum β-lactamases (which should confer a high level of piperacillin-tazobactam resistance) is not observed in clinical practice and why TEM-207 has rarely been detected in clinical isolates., Competing Interests: The authors declare no conflict of interest.

Objectives: Klebsiella aerogenes is a largely understudied opportunistic pathogen that can cause sepsis and lead to high mortality rates. In this study, we reported the occurrence of carbapenem-resistant bla OXA-181 -carrying Klebsiella aerogenes from swine in China and elucidate their genomic characteristics., Methods: A total of 126 samples, including 109 swine fecal swabs, 14 environmental samples, and three feed samples were collected from a pig farm in China. The samples were enriched with LB broth culture and then inoculated into MacConkey agar plates for bacterial isolation. After PCR detection of carbapenemases genes, the bla OXA-181 -carrying isolates were subjected to antimicrobial susceptibility testing, and whole-genome sequence analysis., Results: Four Klebsiella aerogenes isolates carrying the bla OXA-181 gene were obtained from swine faecal samples. All the 4 strains were belonged to ST438. The bla OXA-181 genes were located in IncX3-ColKP3 hybrid plasmids with the core genetic structure of IS26-ΔIS3000-ΔISEcp1-bla OXA-181 -ΔlysR-ΔereA-ΔrepA-ISKpn19-tinR-qnrS1-ΔIS2-IS26, which suggests the potential for horizontal transfer and further dissemination of this resistance gene among Enterobacteriaceae and other sources., Conclusions: This study represents the first instance of OXA-181-producing K. aerogenes being identified from swine faeces in China. It is crucial to maintain continuous monitoring and ongoing attention to the detection of K. aerogenes carrying bla OXA-181 and other resistance genes in pigs., Competing Interests: Conflict of interest We have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Klebsiella aerogenes is an understudied and clinically important pathogen. We therefore investigate its population structure by genome analysis aligned with metadata. We sequence 130 non-duplicated K. aerogenes clinical isolates and identify two inter-patient transmission events. We then retrieve all publicly available K. aerogenes genomes (n = 1,026, accessed by January 1, 2023) and analyze them with our 130 genomes. We develop a core-genome multi-locus sequence-typing scheme. We find that K. aerogenes is a species complex comprising four phylogroups undergoing evolutionary divergence, likely forming three species. We delineate remarkable clonal diversity and identify three worldwide-distributed carbapenemase-encoding clonal clusters, representing high-risk lineages. We uncover that K. aerogenes has an open genome equipped by a large arsenal of antimicrobial resistance genes. We identify two genetic regions specific for K. aerogenes, encoding a type VI secretion system and flagella/chemotaxis for motility, respectively, both contributing to the virulence. These results provide much-needed insights into the population structure and pan-genomes of K. aerogenes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages., Results: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor., Conclusions: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus., (© 2024. The Author(s).)

Objectives: The dissemination of New Delhi metallo-β-lactamase-5 (NDM-5) among various species of Enterobacterales has attracted serious global attention. Here, we characterise the genomic characterisation of bla NDM-5 -IncX3 plasmid (pNDM-KA3) in an ST4 Klebsiella aerogenes (KA3) strain isolated from a neonate with pneumonia., Methods: Antimicrobial susceptibility and multilocus sequence typing was performed for the KA3. The plasmid conjugation assay and plasmid stability of the KA3 (pNDM-KA3) were also analysed. The pNDM-KA3 plasmid was further analysed by whole-genome sequencing and comparative analysis to determine the genetic environment of bla NDM-5 ., Results: The KA3 strain belongs to ST4 and shows high resistance to β-lactam antibiotics, including carbapenems, but is susceptible to ciprofloxacin, amikacin, tigecycline, and colistin. The pNDM-KA3 was successfully transferred to the recipient E. coli J53 and showed strong stability in K. aerogenes. Genomic sequencing revealed that the pNDM-KA3 plasmid was assigned to plasmid incompatibility group X3 with 43367 bp, and a conserved structure sequence of △IS3000-△ISAba125-IS5-bla NDM-5 -ble MBL - trpF-dsbC-IS26 was detected upstream and downstream of the bla NDM-5 gene. Further analysis revealed that insertion sequences mediated the dissemination of bla NDM-5 from other species of Enterobacterales. The pNDM-KA3 showed high similarity to bla NDM-5 -harbouring plasmids in other species of Enterobacterales, with these plasmids carrying genes for replication (repB), partitioning (parA and parB), stability (hns), and conjugative transfer (virB and virD)., Conclusions: Continued monitoring for the dissemination of bla NDM-5 among uncommon Enterobacterales species should be further reinforced., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-β-lactamase (NDM)-1, and NDM-5. The strain harboring bla NDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing bla IMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN 4 plasmid type. However, integrons found in the strains possessing bla IMP-6 , which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae , suggesting the prevalence of identical integrons. Transposons containing bla NDM are similar or identical to the transposon structure of K. aerogenes harboring bla NDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

Klebsiella aerogenes is an emergent pathogen associated with outbreaks of carbapenem-resistant strains. To date, studies focusing on K. aerogenes have been small-scale and/or geographically restricted. Here, we analyzed the epidemiology, resistome, virulome, and plasmidome of this species based on 561 genomes, spanning all continents. Furthermore, we sequenced four new strains from Brazil (mostly from the Amazon region). Dozens of STs occur worldwide, but the pandemic clones ST93 and ST4 have prevailed in several countries. Almost all genomes were clinical, however, most of them did not carry ESBL or carbapenemases, instead, they carried chromosomal alterations (omp36, ampD, ampG, ampR) associated with resistance to β-lactams. Integrons were also identified, presenting gene cassettes not yet reported in this species (blaIMP, blaVIM, blaGES). Considering the virulence loci, the yersiniabactin and colibactin operons were found in the ICEKp10 element, which is disseminated in genomes of several STs, as well as an incomplete salmochelin cluster. In contrast, the aerobactin hypervirulence trait was observed only in one ST432 genome. Plasmids were common, mainly from the ColRNAI replicon, with some carrying resistance genes (mcr, blaTEM, blaNDM, blaIMP, blaKPC, blaVIM) and virulence genes (EAST1, senB). Interestingly, 172 genomes of different STs presented putative plasmids containing the colicin gene., (© 2024. The Author(s).)

2,3-Butanediol (2,3-BDO) is an important gateway molecule for many chemical derivatives. Currently, microbial production is gradually being recognized as a green and sustainable alternative to petrochemical synthesis, but the titer, yield, and productivity of microbial 2,3-BDO remain suboptimal. Here, we used systemic metabolic engineering strategies to debottleneck the 2,3-BDO production in Enterobacter aerogenes. Firstly, the pyruvate metabolic network was reconstructed by deleting genes for by-product synthesis to improve the flux toward 2,3-BDO synthesis, which resulted in a 90% increase of the product titer. Secondly, the 2,3-BDO productivity of the IAM1183-LPCT/D was increased by 55% due to the heterologous expression of DR1558 which boosted cell resistance to abiotic stress. Thirdly, carbon sources were optimized to further improve the yield of target products. The IAM1183-LPCT/D showed the highest titer of 2,3-BDO from sucrose, 20% higher than that from glucose, and the yield of 2,3-BDO reached 0.49 g/g. Finally, the titer of 2,3-BDO of IAM1183-LPCT/D in a 5-L fermenter reached 22.93 g/L, 85% higher than the wild-type strain, and the titer of by-products except ethanol was very low. KEY POINTS: Deletion of five key genes in E. aerogenes improved 2,3-BDO production The titer of 2,3-BDO was increased by 90% by regulating metabolic flux Response regulator DR1558 was expressed to increase 2,3-BDO productivity., (© 2024. The Author(s).)

Klebsiella aerogenes, previously known as Enterobacter aerogenes, is a gram-negative bacterium typically present in the gastrointestinal tract. While numerous studies reported the pathogenicity and drug resistance of this bacterium there remains a lack of comprehensive research on K. aerogenes induced alterations in the host cellular mechanisms. In this study, we identify a previously uncharacterized C. elegans miR-61 that defines an evolutionarily conserved miRNA important for development and innate immunity regulation through Notch and TGF-β signaling pathway. We employed C. elegans wild-type (N2) as well as mutant strains, such as TGF-β (sma-6) and notch-signaling pathway mutants (adm-4 and mir-61). Our results have demonstrated that the K. aerogenes infected mutants exhibited significantly reduced survival rate, reduced pharyngeal pumping, altered swimming and chemotactic behavior. Moreover, K. aerogenes affects the healthspan by increasing ROS level in the mutants. The gene expression analysis revealed that K. aerogenes upregulated egl-30, tph-1 and sod-1 in adm-4, mir-61 mutants not in sma-6. The in-silico analysis indicated an interaction between mir-61 and col-19, which was confirmed by the upregulation of miR-61 expression and the downregulation of col-19 in sma-6, adm-4, and wild-type strains. These findings suggest that C. elegans activates mir-61 and col-19 regulation through the Notch and TGF-β signaling pathway against K. aerogenes infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Balamurugan Krishnaswamy reports was provided by Alagappa University. BALAMURUGAN KRISHNASWAMY reports a relationship with Alagappa University that includes:. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As a concentrated energy source with high added value, hydrogen has great development prospects, with special emphasis on sustainable microbial production as a replacement for traditional fossil fuels. In this study, λ-Red recombination was used to alter the activity of Complex I by single and combined knockout of nuoE, nuoF and nuoG. In addition, the conversion of malic to pyruvic acid was promoted by overexpressing the maeA gene, which could increase the content of NADH and formic acid in the bacterial cells. Compared to the original strain, hydrogen production was 65% higher in the optimized strain IAM1183-EFG/M, in which the flux of the formic acid pathway was increased by 257%, the flux of the NADH pathway was increased by 13%, and the content of metabolites also changed significantly. In further bioreactor, the total hydrogen production of the scale-up IAM1183-EFG/M after 44 h of fermentation was 4.76 L, which increased by 18% compared with the starting strain. This study provides a new direction for future exploration of microbial hydrogen production by combinatorial modification of multiple genes., (© 2023. The Author(s).)

Estradiol decline can result in depressive disorders in females; nevertheless, the causes of this decline are unclear. In this study, we isolated estradiol-degrading Klebsiella aerogenes from the feces of premenopausal females with depression. In mice, gavaging with this strain led to estradiol decline and depression-like behaviors. The gene encoding the estradiol-degrading enzyme in K. aerogenes was identified as 3β-hydroxysteroid dehydrogenase (3β-HSD). Heterologously expressing 3β-HSD resulted in Escherichia coli obtaining the ability to degrade estradiol. Gavaging mice with 3β-HSD-expressing E. coli decreased their serum estradiol levels, causing depression-like behaviors. The prevalence of K. aerogene and 3β-HSD was higher in premenopausal women with depression than in those without depression. These results suggest that the estradiol-degrading bacteria and 3β-HSD enzymes are potential intervention targets for depression treatment in premenopausal women., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Enterobacter spp. and Klebsiella aerogenes are rod-shaped Gram-negative opportunistic pathogens. This study aimed at the molecular and genomic characterization of multidrug resistant Enterobacter spp. and K. aerogenes isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. A total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by Etest were included in this study. Genotypic identification through whole-genome sequencing (WGS) was performed and confirmed in silico . Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multilocus sequence typing (MLST) was used to determine the isolates' clonality. Using the average nucleotide identity (ANI) we identified and confirmed that 47 (80%) isolates were E . hormaechei , 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC 90 of ≥ 32 μg/mL for ertapenem, imipenem, and meropenem. bla NDM-1 (58.5%), bla ACT-16 (54%), and bla OXA-1 (54%) were the most common detected β-lactamases, while bla CTX-M-15 (68%) was the main detected extended-spectrum β-lactamase (ESBL) encoding gene. Chromosomal ampC , carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common among the studied E . hormaechei. : The spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. IMPORTANCE Drug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in health care settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD + was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD + by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD + by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

The prevalence of multidrug-resistant Enterobacter aerogenes strains in UTIs is increasing. Therefore, the purpose of this study was to examine the mechanisms of resistance in Enterobacter aerogenes strains isolated from the urinary tract of infected patients. To achieve this goal, 786 urine samples from Shahrekord, Iran, were collected from June 2019 to February 2020. After isolating and identifying E. aerogenes samples, antibiotic susceptibility testing was done on the strains using Kirby-Bauer's disk diffusion method. The biofilm formation assays were performed to study the link between antibiotic resistance and biofilm formation and virulence genes. As a result, amongst the 786 urine samples, 50 strains were identified as E. aerogenes. The lowest rate of resistance was observed with imipenem (30%). This study also reports that all the strains of E. aerogenes are biofilm producers, with 50% of isolates producing a large amount, 30% a moderate amount, and 20% a small amount of biofilm. 42% were identified in the phenotypic study of ESBLs. In the PCR test, (64%) produced broad-spectrum beta-lactamases. Prevalence of qnrC, qnrB, qnrA, tetA, tet B, acc(3)IIa, acc(2)IIa, ant(2)Ia and Sul1 in strong producing isolates reported 100%, 80.95%,% 58.14, 87.5%, 81.58%, 86.67%, 82.14, 81.48% and 90% respectively. In the statistical analysis based on the chi-square test, a statistically significant relationship was reported between qnrA, qnrB, tetA, tetB, Sul1, ant(2)Ia, ant(3)I, aac(3)II, and biofilm formation. Resistance to cephalothin, ceftriaxone, cefotaxime and ceftazidime were reported 40%, 34%, 30% and 30%, respectively. Out of 50 Enterobacter aerogenes, 32 isolates (64%) were identified in the phenotypic study of ESBLS, prevalence of bla CTX-M , bla TEM and bla SHV reported 30%, 20% and 14% respectively. There is a significant relationship between resistance to ceftriaxone and bla CTX-M. Prevalence of csgA, ybtS, markD, rmpA, csgD and fimH in strong biofilm formation isolates reported 84%, 83.33%, 80%, 80%, 80% and 66% respectively. The chi-square test showed a statistically significant relationship between biofilm production and resistance genes fimH, csgA, csgD, ybtS, and mrkD. The findings of this study indicate that the ability to produce biofilms is associated with the increase of antibiotic resistance and virulence genes. These agents enable bacteria to produce biofilms that ultimately lead to colonization and bacterial survival in the body., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Klebsiella (nee Enterobacter) aerogenes is the first human gut commensal bacterium with a documented sensitivity to the pineal/gastrointestinal hormone melatonin. Exogenous melatonin specifically increases the size of macrocolonies on semisolid agar and synchronizes the circadian clock of K. aerogenes in a concentration dependent manner. However, the mechanisms driving these phenomena are unknown. In this study, we applied RNA sequencing to identify melatonin sensitive transcripts during culture maturation. This work demonstrates that the majority of melatonin sensitive genes are growth stage specific. Melatonin exposure induced differential gene expression of 81 transcripts during exponential growth and 30 during early stationary phase. This indole molecule affects genes related to biofilm formation, fimbria biogenesis, transcriptional regulators, carbohydrate transport and metabolism, phosphotransferase systems (PTS), stress response, metal ion binding and transport. Differential expression of biofilm and fimbria-related genes may be responsible for the observed differences in macrocolony area. These data suggest that melatonin enhances Klebsiella aerogenes host colonization., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In order to invent a porcine gelatine detection device using microbial resources, bacterial enzymes with a preference towards porcine gelatine and their candidate genes were evaluated. Five (n = 5) bacterial strains isolated from hot spring water and wet clay, Malaysia were screened for their gelatinase activity. The gelatinase enzyme was extracted and purified using ammonium sulphate precipitation prior to performing gelatinase assay on porcine, bovine and fish gelatine medium substrates. The G2 strain or Enterobacter aerogenes (Strain EA1) was selected for whole genome sequenced after showing a consistent trend of preference towards porcine gelatine. The gelatinase candidate gene gelEA1_9 was cloned and expressed. Based on one-way analysis of variance (ANOVA) with POST-HOC Duncan test (α = 0.05), the final product of gelEA1_9 was identified as a novel gelatinase. This gelatinase presented no significant difference in activity towards porcine gelatine. Hence, the present study demonstrated an enzyme-substrate interaction for porcine gelatine identification., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bronchoscopes have been linked to outbreaks of nosocomial infections. The phenotypic and genomic profiles of bronchoscope-associated Klebsiella aerogenes isolates are largely unknown. In this work, a total of 358 isolates and 13 isolates were recovered from samples after clinical procedures and samples after decontamination procedures, respectively, over the five months. Antimicrobial susceptibility testing found seven K. aerogenes isolates exhibiting a low-level resistance to antimicrobial agents. Among seven K. aerogenes isolates, we found five sequence types (STs) clustered into three main clades. Collectively, this study described for the first time the phenotypic and genomic characteristics of bronchoscope-associated K. aerogenes ., Competing Interests: Conflict of interest The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication., (© 2021 Fang Huang et al.)

Objectives: The aim of this study was to characterise a tigecycline-resistant bla NDM-9 -bearing Klebsiella aerogenes strain (HNHF1) of chicken origin., Methods: Strain HNHF1 was characterised by phenotypic antimicrobial susceptibility testing, PCR, conjugation assays, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), whole-genome sequencing and bioinformatics analysis., Results: The bla NDM-9 gene was located on an IncHI2 plasmid (pHNHF1_NDM-9) carrying various antimicrobial resistance genes. Moreover, the genetic context ΔISAba125-bla NDM-9 -ble MBL -trpF is similar to other bla NDM -bearing genetic contexts. TA cloning experiments showed that tet(A) variants may play a partial role in high-level tigecycline resistance in HNHF1., Conclusion: This is the first report of a tigecycline-resistant bla NDM-9 -bearing IncHI2 plasmid in a K. aerogenes ST4 isolate of animal origin, which poses a great threat to public health. Further comprehensive surveillance is needed., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet (X) and tmexCD-toprJ , have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet (X) and tmexCD3-toprJ3 along with the carbapenemase gene bla NDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet (X) and tmexCD-toprJ , and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet (X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.

A novel esterase (EstKa) from marine Klebsiella aerogenes was characterized with hydrolytic activity against p-nitrophenyl caprylate (pNPC, C 8 ) under optimum conditions (50 °C and pH 8.5). After two rounds of mutagenesis, two highly potential mutants (I6E9 and L7B11) were obtained with prominent activity, substrate affinity and thermostability. I6E9 (L90Q/P96T) and L7B11 (A37S/Q100L/S133G/R138C/Q156R) were 1.56- and 1.65-fold higher than EstKa in relative catalytic efficiency. The influence of each amino acid on enzyme activity was explored by site-directed mutation. The mutants Pro96Thr and Gln156Arg showed 1.29- and 1.48-fold increase in catalytic efficiency (Kcat/Km) and 54.4 and 36.2% decrease in substrate affinity (Km), respectively. The compound mutant Pro96Thr/Gln156Arg exhibited 68.9% decrease in Km and 1.41-fold increase in Kcat/Km relative to EstKa. Homology model structure analysis revealed that the replacement of Gln by hydrophilic Arg on the esterase surface improved the microenvironment stability and the activity. The replacement of Pro by Thr enabled the esterase enzyme to retain 90% relative activity after 3 h incubation at 45 °C. Structural analysis confirmed that the formation of a hydrogen bond leads to a notable increase of catalytic efficiency under high temperature conditions.

Polymyxins were recently approved for the clinical treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections in China. The aim of this study was to determine the prevalence and molecular mechanisms of polymyxin-resistant CRE prior to the clinical application of polymyxin and to evaluate the potential for emerging polymyxin resistance in China. A total of 504 unique CRE isolates were collected from six tertiary-care hospitals in China between October 2016 and September 2017. All isolates underwent antimicrobial susceptibility testing. Clinical, demographic, antimicrobial exposure and infection data were collected from patients' medical charts. PCR detection, Sanger sequencing and reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) were used to investigate the molecular mechanism of polymyxin resistance. A total 19 (3.8%) polymyxin-resistant isolates were identified, including Klebsiella pneumoniae, Escherichia coli, Klebsiella aerogenes and Enterobacter cloacae. Genetic analysis in K. pneumoniae strains identified insertion sequence (IS) elements (n = 3), a stop codon (n = 1) and gene deletion (n = 2) in mgrB and a pmrB missense mutation (T157P) (n = 1). Two E. coli isolates contained mcr-1 and an E. cloacae strain harboured a frameshift in mgrB. Further transcriptional analysis showed that pmrA, pmrB, pmrC and pmrK were significantly upregulated in polymyxin-resistant isolates. Despite the lack of polymyxin exposure, 3.8% of CRE were resistant to polymyxin in China. Both chromosomal and plasmid-encoded mechanisms were identified. Our study suggests that clinical practice should be alert to pre-existing polymyxin resistance among CRE isolates to avoid further dissemination of polymyxin resistance., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dark fermentative hydrogen production from glucose by Enterobacter aerogenes was studied. The kinetic models of modified Gompertz and Logistic were employed to investigate the progress of hydrogen production. The predicted maximum hydrogen production (H max ) by modified Gompertz and Logistic was 11.92 and 11.28 mL, respectively. The kinetic models of modified Gompertz, Logistic, and Richards were used to study biomass growth in batch experiments. The maximum biomass growth (X max ) by models of modified Gompertz, Logistic, and Richards was 4.90, 4.85, and 4.95 (g L -1 ), respectively. The modified Gompertz was applied to simulate the consumption of glucose where the maximum degraded glucose (S max ) was obtained 19.77 g L -1 . The correlation coefficients of all the models were over 0.97, which illustrate that the models fit the data very well. However, the modified Gompertz model presents higher R 2 and lower RSS and is more appropriate than the other models.

Introduction: Carbapenemase-resistant enterobacteria that produce the bla NDM gene are found worldwide. However, this is the first report of blaNDM in Klebsiella aerogenes in Brazil., Methods: The identification of bacterial species was performed using anautomated system and confirmed by biochemical tests, 16S rRNA gene sequencing, and detection of resistance genes., Results: The clinical isolate showed minimum inhibitory concentration resistance to meropenem and polymyxin B at 8mg/L and 4mg/L, respectively. Only the blaNDM gene was detected., Conclusions: The current report of the blaNDM gene in isolated MDR enterobacteria indicates that this gene can spread silently in a hospital setting.

Rapid and accurate diagnosis of bacterial infections resistant to multiple antibiotics requires development of new bio-sensors for differentiated detection of multiple targets. This work demonstrates 7x multiplexed detection for antibiotic-resistance bacterial screening on an optofluidic platform. We utilize spectrally multiplexed multi-spot excitation for simultaneous detection of nucleic acid strands corresponding to bacterial targets and resistance genes. This is enabled by multi-mode interference (MMI) waveguides integrated in an optofluidic device. We employ a combinatorial three-color labeling scheme for the nucleic acid assays to scale up their multiplexing capability to seven different nucleic acids, representing three species and four resistance genes.

Enterobacter aerogenes was recently renamed Klebsiella aerogenes This study aimed to identify differences in clinical characteristics, outcomes, and bacterial genetics among patients with K. aerogenes versus Enterobacter species bloodstream infections (BSI). We prospectively enrolled patients with K. aerogenes or Enterobacter cloacae complex ( Ecc ) BSI from 2002 to 2015. We performed whole-genome sequencing (WGS) and pan-genome analysis on all bacteria. Overall, 150 patients with K. aerogenes (46/150 [31%]) or Ecc (104/150 [69%]) BSI were enrolled. The two groups had similar baseline characteristics. Neither total in-hospital mortality (13/46 [28%] versus 22/104 [21%]; P  = 0.3) nor attributable in-hospital mortality (9/46 [20%] versus 13/104 [12%]; P  = 0.3) differed between patients with K. aerogenes versus Ecc BSI, respectively. However, poor clinical outcome (death before discharge, recurrent BSI, and/or BSI complication) was higher for K. aerogenes than Ecc BSI (32/46 [70%] versus 42/104 [40%]; P  = 0.001). In a multivariable regression model, K. aerogenes BSI, relative to Ecc BSI, was predictive of poor clinical outcome (odds ratio 3.3; 95% confidence interval 1.4 to 8.1; P  = 0.008). Pan-genome analysis revealed 983 genes in 323 genomic islands unique to K. aerogenes isolates, including putative virulence genes involved in iron acquisition ( n  = 67), fimbriae/pili/flagella production ( n  = 117), and metal homeostasis ( n  = 34). Antibiotic resistance was largely found in Ecc lineage 1, which had a higher rate of multidrug resistant phenotype (23/54 [43%]) relative to all other bacterial isolates (23/96 [24%]; P  = 0.03). K. aerogenes BSI was associated with poor clinical outcomes relative to Ecc BSI. Putative virulence factors in K. aerogenes may account for these differences., (Copyright © 2020 American Society for Microbiology.)

Background: β-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects., Results: A β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co 2+ , Mn 2+ , Zn 2+ , Ba 2+ and Ca 2+ . Man1E showed the highest affinity for Locust bean gum (LBG). The K m and V max values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively., Conclusions: A new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

Introduction: This study examined ceftazidime-avibactam activity against carbapenem-resistant Enterobacterales (CRE) clinical isolates and resistance mechanisms among non-metallo β-lactamase (MBL) producers displaying ceftazidime-avibactam MIC values at 4 mg/L., Methods: CRE isolates (286 of 8161 Enterobacterales) collected in Asia-Pacific, Europe and Latin America during 2016 were screened for carbapenemase genes. Selected isolates were susceptibility tested for ceftazidime-avibactam in the presence or absence of phenylalanine-arginyl β-naphthylamide (PAβN) and polymyxin B nonapeptide (PMBN). Genome sequences were investigated for the integrity of outer membrane protein (OMP) genes and multilocus sequence typing. qRT-PCR assays were conducted to determine expression of acrA, ampC, and OMP genes., Results: Ceftazidime-avibactam inhibited 99.2% of the Enterobacterales, 22 (78.7%) of the 286 CRE and 226 (100%) non-MBL producers. Among carbapenemase producers (85.3%; 244 of 286), the most common gene was bla KPC (76 bla KPC-3 and 46 bla KPC-2 ), followed by bla OXA-48 -like (60 isolates) and bla NDM (37). Ceftazidime-avibactam MIC values at 4 mg/L were noted among 14 Klebsiella pneumoniae (13 carrying bla KPC and 1 bla CTX-M-15 ) mostly from Italy and Brazil and 1 Klebsiella aerogenes overexpressing ampC. PAβN did not significantly decrease ceftazidime-avibactam results, but adding PMBN did significantly decrease the MIC results for the combination. All K. pneumoniae isolates had a premature stop codon at OmpK35 and most isolates had L3 alterations of OmpK36, low expression of this gene, or OmpC disruption (K. aerogenes). Nine K. pneumoniae isolates belonged to clonal complex 258 and displayed intrahospital clonality., Conclusion: Ceftazidime-avibactam is an important addition to the armamentarium against multidrug-resistant organisms, and elevated MIC results for this combination seem to be associated with L3 OmpK36 alterations and β-lactamases able to hydrolyze ceftazidime., (Copyright © 2020. Published by Elsevier B.V.)

Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.

The emergence of bla NDM particularly in Gram-negative bacteria is a burden on the health care system in developing countries. Hence, this study was initiated to screen New Delhi Metallo-β-lactamase (NDM)-producing Gram-negative bacterial strains from neonatal intensive care unit (NICU) of an Indian Hospital. A total of 18 bla NDM -producing isolates were detected in the present study. Out of 18 bla NDM variant isolates, 6 were Klebsiella pneumoniae , 4 Escherichia coli , 2 Enterobacter aerogenes , 1 Acinetobacter lwoffii , 1 Enterobacter cloacae , 3 Acinobacter baumannii, and 1 Cedecea davisae from NICU, showing resistance against all antibiotics, except colistin and polymixin. The transferability of resistance determinants was tested by conjugation. Transfer of bla NDM -producing strains was successful in all 18 strains. In the case of transconjugants, the minimum inhibitory concentration values were found to decrease. The bla NDM -producing isolates contained detectable plasmids of size 66, 38, and 6 kb. Plasmi/d-based replicon typing revealed the incompatibility types Inc (A/C, FIIA, FIC, K, F, W, FIA, P, X, FIB, B/O) in bla NDM -carrying isolates. This study revealed the outbreak of multiple variants of bla NDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). Moreover, other resistance markers, viz. bla OXA-1 , bla C MY-1 , bla VIM-1 , and bla SHV-1 coassociated with bla NDM were also found. In this study, we reported NDM-producing C. davisae as a first report to the best of our knowledge. This study is an attempt to reveal the dissemination of bla NDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Enterobacter aerogenes LU2 was isolated from cow rumen and recognized as a potential succinic acid producer in our previous study. Here, we present the first complete genome sequence of this new, wild strain and report its basic genetic features from a biotechnological perspective. The MinION single-molecule nanopore sequencer supported by the Illumina MiSeq platform yielded a circular 5,062,651 bp chromosome with a GC content of 55% that lacked plasmids. A total of 4,986 genes, including 4,741 protein-coding genes, 22 rRNA-, 86 tRNA-, and 10 ncRNA-encoding genes and 127 pseudogenes, were predicted. The genome features of the studied strain and other Enterobacteriaceae strains were compared. Functional studies on the genome content, metabolic pathways, growth, and carbon transport and utilization were performed. The genomic analysis indicates that succinic acid can be produced by the LU2 strain through the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. Antibiotic resistance genes were determined, and the potential for bacteriocin production was verified. Furthermore, one intact prophage region of length ~31,9 kb, 47 genomic islands (GIs) and many insertion sequences (ISs) as well as tandem repeats (TRs) were identified. No clustered regularly interspaced short palindromic repeats (CRISPRs) were found. Finally, comparative genome analysis with well-known succinic acid producers was conducted. The genome sequence illustrates that the LU2 strain has several desirable traits, which confirm its potential to be a highly efficient platform for the production of bulk chemicals.

Aims: To undertake a prospective analysis of the occurrence of colistin-resistant and extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales colonizing pigs at two farms in Portugal, and to evaluate the putative correlations with usage of different antibiotics., Materials and Methods: One hundred and two faecal samples recovered from two different Portuguese pig farms were screened for polymyxin-resistant and ESBL-positive Enterobacterales. The authors had undertaken a study at one of the farms previously, but the use of colistin has since been banned; zinc oxide and amoxicillin are used as prophylactic and curative drugs, respectively, at this farm. The other farm included in this study used zinc oxide alone., Results: Ninety-three ESBL-producing isolates (62 Escherichia coli, 29 Klebsiella pneumoniae, one Enterobacter aerogenes and one Enterobacter cloacae) and 17 colistin-resistant isolates (12 E. coli, four K. pneumoniae and one E. cloacae) were recovered. Among the ESBL producers, the majority (84%) produced CTX-M-15, while the others produced CTX-M-1 or CTX-M-9. Many different strain and plasmid backgrounds were identified, ruling out a massive dissemination of one major clone. In total, 17 colistin-resistant isolates were recovered, all from the first farm. All produced MCR-1, corresponding to 12 E. coli (10 clones) and three K. pneumoniae (two clones). The MCR-1 producers were all recovered from the farm where colistin had been used 2 years previously., Conclusion: This study showed a surprisingly high rate of CTX-M-15 producers at two Portuguese pig farms. A link was found between antibiotic selective pressure (ß-lactam or polymyxin) and the corresponding resistance rate., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Survival of bacteria on surfaces plays an important role in the cross-contamination of food. Temperature, relative humidity (RH), surface type, and inoculum diluent can affect bacterial survival. This study was conducted to examine how temperature, RH, and diluent affect the survival of Enterobacter aerogenes on stainless steel, polyvinyl chloride, and ceramic tile. Although surface type had little effect on survival, temperature had a clear effect. E. aerogenes survival was highest at 7°C and 15 and 50% RH on all surfaces. Some diluents allowed growth under high RH conditions. Cell populations in distilled water inoculated onto each surface decreased initially compared with populations in 1% phosphate-buffered saline (PBS) and 0.1% peptone broth. At 15 and 50% RH, cell populations in 1% PBS declined more sharply after 120 h than did those 0.1% peptone, but populations in both diluents had similar declines up to 3 weeks. Cell populations in 0.1% peptone had the greatest growth and reached the highest population density (∼8 log CFU/mL). Cell populations in PBS and distilled water increased by ∼2 log CFU/mL. When cells in 0.1% peptone were inoculated onto stainless steel at 100% RH, populations increased to ∼7 log CFU per coupon, whereas cells in 1% PBS increased to ∼5 log CFU per coupon followed by a decline over 3 weeks. DMFit and GInaFiT software modeled inactivation on surfaces at all conditions other than 100% RH at 21°C. These findings have important implications for experiments in which microorganisms are inoculated onto foods or food contact surfaces because the growth observed may be affected more by the inoculum diluent at high or uncontrolled RH than by the type of inoculated surface.

Klebsiella aerogenes is an important pathogen in healthcare-associated infections. Nevertheless, in comparison to other clinically important pathogens, K. aerogenes population structure, genetic diversity, and pathogenicity remain poorly understood. Here, we elucidate K. aerogenes clonal complexes (CCs) and genomic features associated with resistance and virulence. We present a detailed description of the population structure of K. aerogenes based on 97 publicly available genomes by using both multilocus sequence typing and single-nucleotide polymorphisms extracted from the core genome. We also assessed virulence and resistance profiles using Virulence Finder Database and Comprehensive Antibiotic Resistance Database, respectively. We show that K. aerogenes has an open pangenome and a large effective population size, which account for its high genomic diversity and support that negative selection prevents fixation of most deleterious alleles. The population is structured in at least 10 CCs, including two novel ones identified here, CC9 and CC10. The repertoires of resistance genes comprise a high number of antibiotic efflux proteins as well as narrow- and extended-spectrum β-lactamases. Regarding the population structure, we identified two clusters based on virulence profiles because of the presence of the toxin-encoding clb operon and the siderophore production genes, irp and ybt. Notably, CC3 comprises the majority of K. aerogenes isolates associated with hospital outbreaks, emphasizing the importance of constant monitoring of this pathogen. Collectively, our results may provide a foundation for the development of new therapeutic and surveillance strategies worldwide., (© 2019 Federation of European Biochemical Societies.)

Introduction: The objective of this study was to characterize genes of aminoglycoside modifying enzymes (AMEs) in colonizing and infecting isolates of E. aerogenes harboring bla KPC from patients at a public hospital in Recife-PE, Brazil., Methods: We analyzed 29 E. aerogenes clinical isolates resistant to aminoglycosides. AMEs genes were investigated by PCR and sequencing., Results: Colonizing and infecting isolates mainly presented the genetic profiles aac(3)-IIa/aph(3')-VI or ant(2")-IIa/aph(3')-VI. This is the first report of aph(3')-VI in E. aerogenes harboring bla KPC in Brazil., Conclusions: The results highlight the importance in establishing rigorous methods for the surveillance of resistance genes, especially in colonized patients.

Extended spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae remain a critical clinical concern worldwide. The aim of this study was to characterize ESBL-producing K. pneumoniae detected within and between two hospitals in uMgungundlovu district, South Africa, using whole genome sequencing (WGS). An observational period prevalence study on antibiotic-resistant ESKAPE (i.e. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) bacteria was carried out in hospitalized patients during a two-month period in 2017. Rectal swabs and clinical specimens were collected from patients hospitalized and were screened for ESBL-producing, Gram-negative ESKAPE bacteria using cefotaxime-containing MacConkey agar and ESBL combination disk tests. Nine confirmed ESBL-K. pneumoniae isolated from six patients and two hospitals were whole genome sequenced using an Illumina MiSeq platform. Genome sequences were screened for presence of integrons, insertion sequences, plasmid replicons, CRISPR regions, resistance genes and virulence genes using different software tools. Of the 159 resistant Gram-negative isolates collected, 31 (19.50%) were ESBL-producers, of which, nine (29.03%) were ESBL-K. pneumoniae. The nine K. pneumoniae isolates harboured several β-lactamase genes, including bla CTX-M-15 , bla TEM-1b , bla SHV-1 , bla OXA-1 concomitantly with many other resistance genes e.g. acc(6')-lb-cr, aadAI6, oqxA and oqxB that confer resistance to aminoglycosides and/or fluoroquinolones, respectively. Three replicon plasmid types were detected in both clinical and carriage isolates, namely ColRNAI, IncFIB(K), IncF(II). Sequence type ST152 was confirmed in two patients (one carriage isolate detected on admission and one isolate implicated in infection) in one hospital. In contrast, ST983 was confirmed in a clinical and a carriage isolate of two patients in two different hospitals. Our data indicate introduction of ESBL-producing K. pneumoniae isolates into hospitals from the community. We also found evidence of nosocomial transmission within a hospital and transmission between different hospitals. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated cas3 genes were further detected in two of the nine ESBL-KP isolates. This study showed that both district and tertiary hospital in uMgungundlovu District were reservoirs for several resistance determinants and highlighted the necessity to efficiently and routinely screen patients, particularly those receiving extensive antibiotic treatment and long-term hospitalization stay. It also reinforced the importance of infection, prevention and control measures to reduce the dissemination of antibiotic resistance within the hospital referral system in this district.

Objective: The main focus of this study was to evaluate the antimicrobial susceptibility profiles of a number of human clinical isolates of Enterobacter aerogenes isolates and to explore the effects of selected chemosensitisers on reversal of the resistant phenotype of these isolates., Methods: This study design was accomplished by: (i) characterising several multidrug-resistant (MDR) E. aerogenes clinical isolates; (ii) evaluating the contribution of target gene mutations to the resistance phenotype, focusing on fluoroquinolones and chloramphenicol only; (iii) evaluating the contribution of membrane permeability and efflux to the MDR phenotype; (iv) assessing the combined action of selected antimicrobials and chemosensitisers in order to identify combinations with synergistic effects able to reduce the minimum inhibitory concentration (MIC); and (v) understanding how these combinations can modulate the permeability or efflux of these isolates., Results: Resistance to ciprofloxacin could not be totally reversed owing to pre-existing mutations in target genes. Chloramphenicol susceptibility was efficiently restored by the addition of the selected chemosensitisers. From the modulation kinetics it was clear that phenothiazines were able to increase the accumulation of Hoechst dye., Conclusions: Modulation of permeability and efflux in the presence of chemosensitisers can help us to propose more appropriate chemotherapeutic combinations that can set the model to be used in the treatment of these and other MDR infections., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Background: Bacteria with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity could decrease the ethylene level, confer resistance of plant, and stimulate plant growth under biotic and abiotic stress conditions., Results: Plant growth-promoting rhizobacteria (PGPR) strains Enterobacter aerogenes (LJL-5) and Pseudomonas aeruginosa (LJL-13) were obtained from the rhizosphere of alfalfa grown under saline-alkali conditions. The ACC deaminase activity of E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) was approximately 2-5 µmol mg -1  h -1 . indole acetic acid synthesis was increased with the increasing concentration of l-tryptophan. Siderophore production and phosphate solubilization in Ps. aeruginosa (LJL-13) were higher than those in E. aerogenes (LJL-5). Compared to the non-inoculated seedlings (1.31 ng mL -1  h -1 ), inoculated alfalfa seedlings with E. aerogenes (LJL-5) (0.90 ng mL -1  h -1 ) and Ps. aeruginosa (LJL-13) (0.78 ng mL -1  h -1 ) emitted lower levels of ethylene. Under saline-alkali conditions in the greenhouse, inoculation with E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) increased the biomass, soil and plant analyzer development (SPAD), and P content of alfalfa plants, and also induced the activity of antioxidant enzymes (superoxide dismutase, peroxidase and catalase), promoted the accumulation of antioxidant substances and removed various harmful substances. Under saline-alkali conditions in the field (2012, 2013, and 2014), inoculation of alfalfa with E. aerogenes (LJL-5) and Ps. aeruginosa (LJL-13) significantly increased the shoot height, fresh and dry weights, yield and crude protein content of alfalfa plants, but decreased the fiber content., Conclusion: Two PGPR strains were isolated from the rhizosphere of alfalfa in saline-alkali conditions. Both strains could promote alfalfa growth in saline-alkali soil, and could be used as biofertilizer to promote plant growth under stress and reduce environmental pollution caused by fertilizers simultaneously. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Purpose: The more frequent reports of carbapenem-resistant Enterobacteriaceae have raised the alarm for public health. Apart from the production of carbapenemases, deficiency (decreased or loss of expression) of outer membrane proteins (OMPs) has been proposed as a potentially important mechanism of carbapenem resistance. The aim of the present study was to evaluate the contribution of the major OMPs to carbapenem resistance in Enterobacter aerogenes (CREA) isolates and also investigate the role of small RNAs (sRNAs) in inducing porin-associated permeability defects., Materials and Methods: The differential expression of OMPs was analyzed in four clinical CREA isolates. omp35 and omp36 genes were further investigated by whole-genome sequencing, induction of meropenem resistance, sRNA overexpression, OMP complementation assays, and reverse transcription-quantitative PCR., Results: All four isolates examined were deficient in omp35 and omp36. Functional restoration of these two genes confirmed their contribution to carbapenem resistance. The meropenem induction assay further revealed that porin deficiency plays a role in carbapenem resistance under antibiotic selection pressure. Single-point mutations in omp36 leading to premature stop codons were detected in two of the isolates. Elevated expression levels of the sRNAs micF and micC were detected in the other two porin-deficient isolates, which were predicted to be potential porin regulators from whole-genome sequencing. Overexpression of micF and micC downregulated the expression of Omp35 and Omp36, respectively., Conclusions: Porin deficiency plays an important role in carbapenem resistance among clinical E. aerogenes isolates under regulation of the sRNAs micC and micF. Furthermore, overexpression of micC and micF had a minor to no impact on carbapenem minimum inhibitory concentrations, and thus, the regulatory mechanism is likely to be complex.

Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several β-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Objectives: The draft genome sequences of two Enterobacter aerogenes strains (HN503E2II and PN108E5IIB) isolated from two Cameroonian abattoirs are reported., Methods: Bacterial genomic DNA of the two isolates was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using CLC Genomics Workbench and SPAdes. The assembled contigs were annotated, and antibiotic resistance genes, virulence factors and plasmids were identified., Results: Whole-genome sequencing revealed that both strains were similar, with genomes of 4878638bp and 4794257bp, encoding several resistance genes associated with resistance to β-lactams, fluoroquinolones, aminoglycosides, fosfomycin, phenicols, sulphonamides, trimethoprim, macrolides and tetracycline. In silico analysis also revealed chromosomal integration of one plasmid in the genome of PN108E5IIB., Conclusion: The genome sequences reported here will provide useful information for a better understanding of the genetic structure of E. aerogenes in Africa., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate- co -3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas . In vivo expression of the putative PHA synthase gene ( phaC Pf ) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli ( phaC Pf + , pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant ( phaC Pf + ) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaC Pf , suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Background: IMP-4 class B metallo-β-lactamase-producing Enterobacteriaceae are resistant to carbapenems. The aim of this study was to characterize of IMP-4 metallo-β-lactamase (MBL)-producing Enterobacter aerogenes clinical isolate., Methods: IMP-4 MBL-producing E. aerogenes clinical isolate was collected from a Korean Hospital in 2017. Antimicrobial susceptibility was determined by disk diffusion methods. Further, minimum inhibitory concentrations of β-lactams were determined by Etest. Detection of bla genes was performed by PCR. The genetic organization of class 1 integron carrying the MBL gene cassette was investigated by PCR mapping and sequencing., Results: E. aerogenes strain YN170501 exhibited resistance to penicillins, cephalosporins, and carbapenems and was susceptible to monobactam, aminoglycosides, fluoroquinolone, tigecycline, and trimethoprim-sulfamethoxazole. The blaIMP-4 gene was located in class 1 integron., Conclusions: The blaIMP-4 gene has never been reported in Enterobacter aerogenes clinical isolate from Korea.

We investigated the colistin resistance rate among 356 Enterobacter spp. clinical isolates from eight hospitals in Korea. Antibiotic susceptibility testing was performed by broth microdilution. While 51 of 213 (23.9%) Enterobacter cloacae isolates were colistin-resistant, only six of 143 (4.2%) E. aerogenes isolates showed resistance. We also identified the skip well phenotype in eight E. cloacae and three E. aerogenes isolates. Multilocus sequence typing for E. cloacae and randomly amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus PCR for E. aerogenes revealed that clonal spreading of colistin-resistant and skip well Enterobacter spp. isolates had not occurred. In vitro time-kill assays were performed with three colistin-resistant, three skip well, and two colistin-susceptible isolates of E. cloacae and E. aerogenes. Inconsistent results were observed among isolates with skip well phenotypes; while some were eradicated by 2 mg/L colistin, others were not. This suggests that skip well isolates have differentiated into different categories. As the high rates of colistin resistance in E. cloacae detected are of clinical concern, continuous monitoring is warranted. In addition, the clinical implications and mechanisms of the skip well phenotype should be investigated to ensure the appropriate use of colistin against Enterobacter infections.

Enterobacter aerogenes, a gram-negative, rod-shaped bacterium, is an effective producer of succinate from glucose via the reductive tricarboxylic acid cycle under anaerobic conditions. However, to date, succinate-exporter genes have not been identified in E. aerogenes, although succinate exporters have a large impact on fermentative succinate production. Recently, we genetically identified yjjP and yjjB, as genes encoding a succinate transporter in Escherichia coli. Evaluation of the yjjPB homologs in E. aerogenes (EayjjPB genes) showed that succinate accumulation increased from 4.1 g L -1 to 9.1 g L -1 when the EayjjPB genes were expressed under aerobic conditions. Under anaerobic conditions, succinate yield increased from 53% to 60% by EayjjPB expression and decreased to 48% by deletion of EayjjPB. Furthermore, the production levels of fumarate and malate, which are intermediates of the succinate-biosynthesis pathway, were also increased by EayjjPB expression. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both EaYjjP and EaYjjB are required for the restoration of succinate production. Taken together, these results suggest that EaYjjPB function as a dicarboxylate transporter in E. aerogenes and that the products of both genes are required for dicarboxylate transport., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)